The 5-Hydroxytryptamine3 (5-HT3) receptor, which may be composed of homomers of A subunits or heteromers of A and B subunits, is the least studied member of the nicotinic acetylcholine receptor family of ligand-gated ion channels. In addition to its role in mediating synaptic transmission in the nervous system, it also regulates gastrointestinal motility and the vomiting reflex. The recently published crystal structure of the acetylcholine binding protein has identified the generalized structure of the N-terminal domains of this superfamily, but the precise three-dimensional configuration of the ligand recognition site and the residues involved in mediating ion channel activation are unknown. The goal of this proposal is to map amino acid residues that contribute to ligand recognition and model their spatial configuration. Mouse and human 5-HT3A receptors possess 84 percent identity at the amino acid level, yet have differential sensitivities to numerous drugs that bind to the ligand recognition site, such as d-tubocurarine (curare) and 3-(2-hydroxy, 4-methoxy-benzylidene)-anabaseine (2-OHMBA). The addition of the B subunit to the A subunit further alters potency of these compounds. Mouse-human and human-mouse chimeras will be constructed and expressed in Xenopus oocytes. Domains responsible for the change in drug action will be assessed with two-electrode voltage clamp electrophysiological recordings. Individual amino acids will then be identified for their roles in conferring sensitivity. Structure-activity relationships of key moieties in the substituted 3-benzylideneanabaseine analogs with identified residues will be assessed. Thermodynamic mutant cycle analysis will pinpoint the specific point of contact of the key drug moiety with the identified amino acid residue in the ligand binding domain. 5-HT3A receptors with C-terminal hexa-histidine tags will be expressed and purified. Receptor will then be photolabeled with [3H]-5-HT and [14C]-2-OHMBA, with and without azido side-chains, and radiolabeled residues will be identified through sequencing. Amino acids identified with photoaffinity labeling and electrophysiological experiments, along with the structures of 5-HT, curare, and benzylidene-anabaseine analogs, will serve as a template for molecular modeling studies of the agonist-recognition site.